In academia and pharmaceutical industry great resources are spent on discovery and development of monoclonal antibodies for target-based therapies. Among the available technologies developed thus far, high-throughput techniques for screening antibody libraries have enabled the identification of new candidate molecules and the fast optimization of pre-selected binders by affinity maturation.
As the identification of new candidate molecules is to a great extend technology driven, the invention of new powerful screening technologies has proven to be one critical part in an overall strategy to further accelerate the process of antibody discovery and development. Several in vitro display technologies have emerged since the advent of phage display technologies in the mid-eighties and its application for antibody display. Next to phage display, there are four main display technologies referred to cell display, ribosomal display, mRNA display and DNA display, with phage display being the most established one.
Phage display is currently the most widespread method for the display and selection of large collections of antibodies and for the further engineering of selected antibodies. Antibodies are usually displayed in what is known as the ‘monovalent’ format, in which the antibody-coat protein fusion gene is carried on a phagemid vector and display is performed by infecting the phagemid-carrying bacteria with a helper phage. This format, also known as the 3×3 format, is mostly preferred because constructing libraries in phagemid vectors offer higher transformation efficiency of phagemid vectors compared with phage vectors, as it provides for the selection of the highest affinity binders, which are not skewed by avidity effects (Saggy et al. (2012) Protein Eng. Des. Sel. 25, 539-549).
The most successful applications of phage antibody display include for example de novo isolation of high-affinity human antibodies from non-immune and synthetic libraries, including antibodies against self-antigens, the generation of high affinity antibodies with picomolar affinity by in vitro affinity maturation and the discovery of antibodies with unique properties from non-immune and immune libraries from animal or human donors (Hogenboom (2005) Nat. Biotech 23, 1105-1116).
Despite the advantages of phage antibody display this technology also has disadvantages which limit its use: In E. coli efficient secretion of functional antibody fragments into the periplasmic space typically requires co-expression of chaperones and isomerases to prevent misfolding and aggregation of antibody fragments due to the limited secretion capacity (Bothmann and Plückthun (2000), J. Biol. Chem. Vol. 275 (22), 17100-17105). In addition, there appears to be a biological selection against odd numbers of cysteines, runs of positive charges, and certain residues at fixed positions within the displayed peptide which consequently results in an inherently biased selection of antibody fragments.
The second most frequently used technology is yeast display, which is a robust technology to select and engineer antibody-fragments from combinatorial libraries. Yeast display technologies are advantageous for the expression of oligomeric molecules, such as e.g. full-length IgG immunoglobulins, as the antibodies have to pass the eukaryotic secretion pathway compared to bacterially expressed antibodies, which results in an overall larger number of correctly folded immunoglobulins.
Yeast display utilizes the presence of several naturally occurring cell wall anchored proteins, which can be used to target heterologous proteins to the outermost cell surface via attachment of a C-terminal glycosylphosphatidylinositol attachment signal, commonly referred to as GPI anchor. Initially, yeast display relied on the genetic fusion of antibody-coding DNA sequences in-frame with the sequence of a yeast cell wall mannoprotein (Doerner et al. (2014), FEBS Letters 588, 278-287). The protein repertoire, which is used for surface display was expanded and now includes for example a-agglutinin, Flo1p and α-agglutinin. Of those, the a-agglutinin employing system is the most frequently used.
A-agglutinin is one of the two mating type specific agglutinins that mediate cell-cell contact during mating of appropriate yeast cells. It is formed by one core-subunit Aga1p, which is linked to a smaller binding-subunit Aga2p through two disulfide bridges. Due to the GPI-attachment-signal of Aga1p the core-subunit covalently anchors the Aga complex to the cell wall. The modular structure of a-agglutinin furthermore enables the fusion of the heterologous protein to be displayed to the C- or N-terminus of Aga2p compared to single-unit GPI-anchored proteins that only allow N-terminal fusion of heterologous proteins, due to the required C-terminal GPI-attachment signal. Flo1p-based systems differ in that they can attach and immobilize heterologous proteins non-covalently via fusion to the N-terminal flocculation functional domain that is believed to bind to carbohydrate units on the cell-surface.
The overexpression of chromosomally encoded AGA1 and the episomally encoded AGA2-fusion proteins is typically driven by the inducible Gal10-promoter, which accounts for stoichiometric expression levels of both subunits which associate in the endoplasmatic reticulum. Galactose-induced expression results in the display of approximately 104-105 copies of the fusion-protein on the surface of a host cell (Doerner et al. (2014), FEBS Letters 588, 278-287, Boder and Witrup (1997) Nat. Biotechnol. 15, 553-557). Detection of surface-exposed fusion proteins occurs by virtue of epitope tags or by means of its activity, which in case of an antibody is its binding-affinity to a soluble antigen. The detection is typically carried out with the respective biotinylated antigen and a secondary reagent such as streptavidin-conjugated fluorophores, or an otherwise labelled antigen.
In one approach yeast display was modified which is known as secretion-and-capture cell-surface display for selection of target-binding proteins (SECANT™, Rakestraw et al. (2011) Protein Eng Des Sel. June; 24(6):525-30). This technology was successfully used to display full-length immunoglobulin G (IgG) antibodies on the surface of yeast cells. In the SECANT™ technology the protein of interest (POI) is genetically fused to the small biotin acceptor peptide (BAP) followed by a TEV protease cleavage site to facilitate purification. The TEV-BAP peptide may be fused to the N- or C-terminus of the POI. On the end of the POI opposite to the TEV-BAP tag, the POI is genetically fused to a tag, which is typically a FLAG-tag, whereby the entire BAP, POI and FLAG-tag gene is located 3′ to a yeast secretory signal, typically the engineered aMFpp8 leader sequence, invertase leader sequence, or a synthetic leader sequence, which is followed by Kex2 proteotlytic cleavage site (Rakestraw et al. (2011) Protein Eng Des Sel. June; 24(6):525-30). For the selection of a POI, the gene of the POI is expressed as an N- or C-terminal fusion to a BAP, which is co-expressed with BirA biotin ligase and chaperones. The BirA biotin ligase biotinylates the BAP tag on the POI. Upon secretion, the POI is then bound by surface-localized avidin and can be labeled with a fluorophore-tagged anti-epitope antibodies or fluorophore-tagged antigen for subsequent detection and selection.
While the SECANT technology allows the secretion and selection of complex molecules such as IgG immunoglobulins, this technology still requires the genetic modification of a POI and co-expression of a biotin ligase, which adds additional steps in the screening and selecting procedure.
The continued demand for yeast display and in particular for the display of complex molecules and its use in antibody identification and maturation, there is also a continued requirement to reduce the costs and time associated with the screening procedure to identify new antibody candidates.
It is thus an objective of the present invention to provide a method which allows display of complex molecules on the surface of host cells for identification of a protein of interest, without the requirement for genetically encoded anchor proteins or intracellular antibody modification.